Fig 2: Analysis of the neuroregenerative and neuroprotective effects of clinical MSCs cultured in MSCGM or NLRM. a Neuroregeneration assay in spinal cord neuron (seeded in transwell inserts) and MSCs (seeded in culture wells) cocultures incubated for 5 days. The density of GAP-43-positive immunoreactivity in the bottom side of the transwell following scrape injury was analyzed and is shown in the bar chart. Representative GAP-43-positive micrographs are shown (bar = 200 µm). b Neuroprotection test in spinal cord neuron cultures and MSC cocultures after H2O2 (120 µM for one day) treatment. The apoptotic cells in neuron cultures after coculture with MSCs and H2O2 treatment were processed for TUNEL staining, and representative micrographs are shown (bar = 200 µm). The bar chart shows the quantification of the TUNEL-positive cells. Data are presented as the mean ± SEM. **p < 0.01 compared with the control group using one-way ANOVA with Tukey’s multiple comparisons test

Fig 3: Inhibition of Chk2 promotes DC axon regeneration in vivo.(A) Western blot and (B) densitometry to show that Chk2i significantly suppresses pChk2T68 and pChk2T383 levels after DC injury without affecting pChk1 levels. (C) Many GAP43+ axons [green (DAPI+ nuclei = blue)] were observed in DC + Chk2i regenerating through the lesion site and into the rostral cord (boxed region = high power view of GAP43+ axons in the rostral cord) despite the presence of a large cavity (#), while few GAP43+ axons were present beyond the lesion site in DC + vehicle– and DC + Chk1i–treated spinal cords. GAP43 staining [C (i)] was confirmed with biotynilated dextran amine (BDA) tracing of the same axons [C (ii)]. (D) Quantification of the number of GAP43+ axons at distances caudal and rostral to the lesion site showing significant proportions of axons regenerating up to 6 mm beyond the lesion epicenter. Scale bars in (C), 200 µm. **P = 0.0012 and ***P = 0.0001, ANOVA with Dunnett’s post hoc test. n = 6 nerves per treatment, three independent repeats (total, n = 18 nerves per treatment). (E) Spike 2 software–processed CAP traces from representative sham controls, DC + vehicle–, DC + Chk1i–, and DC + Chk2i–treated rats at 6 weeks after DC injury and treatment. Dorsal hemisection at the end of recording ablated all CAP traces. (F) Negative CAP amplitudes and (G) CAP area at different stimulation intensities were both significantly attenuated in DC + vehicle– and DC + Chk1i–treated rats but were restored in DC + Chk2i–treated rats [P = 0.0001, one-way ANOVA with Dunnett’s post hoc test (main effect)]. (H) Mean tape sensing/removal times and (I) mean error ratio to show the number of slips versus total steps are both restored to normal 3 weeks after treatment with Chk2i [***P = 0.0001, independent sample t test (DC + vehicle versus DC + Chk2i at 3 weeks)], while a significant deficit remains in DC + vehicle– and DC + Chk1i–treated rats (#P = 0.00014, generalized linear mixed models; ##P = 0.00011, linear mixed models over the whole 6 weeks). n = 6 rats per treatment, three independent repeats (total, n = 18 rats per treatment).

Fig 4: Comparative characterization of commercial mesenchymal stem cells (MSCs) cultured in MSCGM or NRLM and the function of the conditioned medium (CM). a MSC characteristics, including morphology (phase-contrast micrograph, upper panel), cell surface expression of CD markers (by flow cytometry, middle panel), and adipocyte differentiation (oil-positive signal after oil staining, lower panel), are shown. b The effect of MSCGM-CM and NRLM-CM on the extension of regenerated neurites (GAP-43-positive neurite). c Quantitative results from b. d The effect of MSCGM-CM and NRLM-CM on LPS stimulation in BV2 microglia. Nitrite release in the medium was measured after microglia were treated with LPS in the presence of CM (0–2% v/v). Data represent the mean ± SEM. *p < 0.05 indicates statistical significance using one-way ANOVA with Tukey’s multiple comparisons test

Fig 5: Chk2 inhibition using prexasertib prevents RGC apoptosis and stimulates neurite outgrowth/axon regeneration after 4 days in vitro and 24 days after ONC in vivo.(A) Preoptimized concentration of prexasertib (Prex) in culture at 4 days significantly enhanced RGC survival compared with control NBA, positive control CNTF (preoptimized), or Chk1i. Prexasertib also enhanced neurite outgrowth, increasing the (B) % RGC with neurites and the (C) mean neurite length compared to all other treatment groups. (D) Representative images from RGCs treated with vehicle, Chk1i, and prexasertib. Scale bars, 100 µm. n = 3 wells per treatment per test, three independent repeats (total, n = 9 wells per treatment per test). (E) Representative images of FG-labeled RGCs in retinal wholemounts at 24 days after ONC in vivo and (F) quantification show that prexasertib significantly protected RGCs from death. Scale bars in (E), 50 µm. (G) Representative images of longitudinal sections of optic nerves at 24 days after ONC stained for GAP43 from ONC + vehicle, ONC + Chk1i, and ONC + prexasertib and (H) quantification to show that prexasertib significantly enhanced RGC axon regeneration through the lesion site (*) and into the distal optic nerve segment [n = 6 nerves per condition, three independent repeats (total, n = 18 nerves per condition)]. P = 0.0001, ANOVA with Dunnett’s post hoc test. Scale bars in (G), 200 µm. (I) The normal ERG trace in intact rats were ablated in ONC + vehicle and ONC + Chk1i treatment, but a significant ERG trace is restored in ONC + prexasertib–treated rats. (J) Mean pSTR amplitudes demonstrate that prexasertib restored significant visual function after ONC. P = 0.0001, ANOVA with Dunnett’s post hoc test. Note: In (H), ONC + vehicle and ONC + Chk1 values are similar and hence appear superimposed.